X‐ray Diffraction at Synchrotron Light Sources


The elucidation of biological structure and function for macromolecules and their complexes is undertaken extensively using X‐ray diffraction. Synchrotron X‐radiation is widely used, due to its exceptional X‐ray brightness and tunability properties, in protein crystallography, fibre diffraction and solution scattering. The synchrotron is also now increasingly used in chemical crystallography of smaller molecules which obviously are very important with respect to all the various ligands that bind to biomolecules; a special feature is the use of the X‐ray intensity to overcome small crystal volume, in ways entirely analogous to biomolecular microcrystals. Techniques that are complementary to crystallography are electron microscopy (EM) and NMR spectroscopy for structure determination and the study of dynamics. Spectral monitoring of samples is undertaken in time‐resolved crystallography to identify structural intermediates and to monitor for radiation damage of biomolecules including metal atom oxidation state changes.

Key Concepts:

  • Structure and function relationships are key to understanding of biomolecules and their roles in life processes, the details of which are revealed at the atomic level by X‐ray diffraction.

  • Conformational changes and information on the dynamics of biomolecules can be determined from several structures in comparison or directly via freeze trapped or time‐resolved structures that have been determined by diffraction studies.

  • Multi‐macromolecular complexes being large and often flexible are especially challenging for crystallisation of sufficiently ordered crystals.

  • Synchrotron radiation sources provide intense and tunable X‐rays that are widely used properties but also the properties of the polarisation and the pulsed nature of these sources can be harnessed in special experiments.

  • Free Electron Lasers (FELs) and X‐ray FELs considerably extend the peak brightnesses available by upto 10 orders of magnitude and shorten the pulse lengths to a few tens of femtoseconds (from sub‐nanoseconds in synchrotrons).

  • Solving a crystal structure has presented special obstacles since the discovery of X‐ray diffraction but synchrotron radiation has opened up new possibilities based on anomalous X‐ray scattering.

  • Pharmaceutical design is possible based on a knowledge of the three‐dimensional structure of a protein receptor site for a ligand, an approach known as structure‐based drug discovery and optimisation.

  • Proteins and their ligands are studied extensively by crystallography either together or separately with the techniques of macromolecular and chemical crystallography, respectively.

Keywords: protein crystallography; fibre diffraction; solution scattering; structural techniques

Figure 1.

A schematic diagram of the layout of a synchrotron radiation (SR) source. In practice, SR storage rings are much larger and complex, having many more machine ‘cells’ than the four shown here. At the European SR Facility (ESRF) in Grenoble, for example, the third‐generation high‐brightness source is 840 m in circumference and has 32 cells. The R.F. (radio frequency) cavity replenishes the energy to the electrons that is lost to the light energy emitted.

Figure 2.

A polychromatic Laue diffraction pattern from a protein crystal with spots colour‐coded for wavelength. Short wavelengths in blue and then through the rainbow colours to red at the longest wavelengths. Actual wavelength bandpass used in this simulation is 0.5–2.5 Å. Reproduced with the permission of Acta Crystallographica.

Figure 3.

(a) Electron density map image of a protein crystal. (b) Ribbon diagram of the same protein crystal to the same scale and the same view. The protein is the tetramer of concanavalin A from jack beans with glucoside in atom‐by‐atom format and metal atoms also shown as individual spheres. Concanavalin A–sugar complexes are typical of the proteins studied to high resolution at a second‐generation SR source (in this case Daresbury SRS), that is around 100 kDa in the asymmetric unit of the crystal.

Figure 4.

A monochromatic rotating crystal diffraction pattern recorded from a human rhinovirus crystal at the Cornell High Energy SR Source (CHESS), USA. Rhinovirus was the first mammalian virus structure to be solved (in 1985). Reproduced with the permission of Professor MG Rossmann, Purdue University, USA and Acta Crystallographica.


Further Reading

Chayen NE, Helliwell JR and Snell EH (2010) Macromolecular Crystallization and Crystal Perfection. Oxford University Press. International Union of Crystallography Monographs on Crystallography ISBN‐10: 0199213259.

Cianci M, Helliwell JR, Helliwell M et al. (2005) Anomalous scattering in structural chemistry and biology. Crystallography Reviews 11: 245–335.

Helliwell JR (1992) Macromolecular Crystallography with Synchrotron Radiation. Cambridge University Press. Published in paperback 2005 DOI: 10.2277/0521544041.

Hendrickson W, Horton JR and LeMaster DM (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three‐dimensional structure. EMBO Journal 9: 1665–1672.

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Helliwell, John R(Oct 2010) X‐ray Diffraction at Synchrotron Light Sources. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003109.pub2]